1. Field of the Invention
The present invention relates to a column packing material and a process for producing the same. More specifically, it relates to an improvement in the modification step of the active group.
2. Description of the Related Art
Currently, columns packed with packing materials are used for a separation of various liquid samples, and a liquid chromatography, particularly a high performance liquid chromatography (HPLC), is frequently used for a separation and analysis of a sample of a mixture of various substances such as serum, etc., and industrially, column packing materials are applied for a separation and extraction of specific components.
In the prior art, when drugs or metabolites in the biological components containing a large amount of proteinaceous components such as serum are to be quantitated by HPLC, to remove difficulties caused by an adsorption of proteins onto the packing material, a pre-treatment such as protein removal is required.
This pre-treatment operation, however, requires much time and labor, and has a problem in that it lowers the precision of the analysis.
Accordingly, column packing materials have been developed which allow a direct injection of a sample containing proteinaceous components, without the need for the protein removal operations, to thereby enable a separation of the various components contained in the sample.
These improved column packing materials employ a porous glass or silica gel as the support, and have different properties imparted inside or outside of the micro pores thereof.
Due to the use of these packing materials, since proteins in a serum (e.g., albumin or globulin) are macromolecules, they do not penetrate the micro pores and are not adsorbed on the hydrophilic outer surface (i.e., pore outer surface), but pass the column as is, but molecules of drugs with relatively smaller sizes are adsorbed on the hydrophilic inner surface (i.e., pore inner surface), to be thus separated in the serum.
Examples of such packing materials are those described in Japanese Unexamined Patent Publication (Kokai) No. 60-56256. In this packing material, a protein is coated on the outer surface of a silica chemically bound with octadecyl (ODS) groups. The coated protein comprises a bovine serum albumin or house rabbit plasma protein, and a packing material is obtained by adsorbing the protein onto the ODS-bound silica and modifying same.
Of the packing materials as mentioned above, however, problems arise with regard to the durability and separation ability of the ODS silica packing material coated with a protein; namely, the adsorbed and modified protein may be eluted when used over a long term, and a column having a high separation efficiency can not be obtained.
To solve these problems, a method has been developed whereby a column as described in Japanese Unexamined Patent Publications (Kokai) Nos. 61-65159 and 1-123145 is obtained, by:
(1) introducing hydrophilic groups into the inner surface and the outer surface of a porous support;
(2) cleaving only the hydrophilic groups on the outer surface by using an enzyme which is itself a macromolecule and cannot penetrate the micro pores of silica; and
(3) introducing hydrophilic groups into the outer surface.
More specifically, in the method described in Japanese Unexamined Patent Publication (Kokai) No. 61-65159, by using a porous silica having glycerylpropyl groups introduced therein as the starting material, and an oligopeptide (e.g., glycylphenylalanyl-phenylalanine) bound thereto through carbonyldiimidazole and hydrolyzed by using carboxypeptidase A, which is a protein hydrolase, the phenylalanine side chain is cleaved on the outer surface.
As a result, glycyl-phenylalanyl-phenylalanine remains as the hydrophobic ligand on the inner surface of the filler, and the outer surface becomes a hydrophilic glycyl-glycerylpropyl group.
Further, in the method described in Japanese Unexamined Patent Publication (Kokai) No. 1-123145, a porous silica having an aminopropyl group introduced therein is used as the starting material, and reacted with octanoyl chloride in the presence of triethylamine to introduce a hydrophobic group through the amide bond, the acyl group on the outer surface is then hydrolyzed with polymyxin acylase, and the amino groups on the outer surface are made hydrophilic by carrying out the reaction with glycidol.
Nevertheless, in the method described in Japanese Unexamined Patent Publication (Kokai) No. 61-65159 or 1-123145, since an enzymatic reaction is utilized the steps are complicated, and further, the characteristics of the packing material are apt to be varied.
Also, these packing materials have problems in that the pH of the eluent is limited to a narrow range, and that it is difficult to obtain stable and reliable measurement results.